| Summary: | A three-year survey was conducted to estimate the incidence of grapevine trunk diseases (GTDs) in Greece and identify fungi associated with the disease complex. In total, 310 vineyards in different geographical regions in northern, central, and southern Greece were surveyed, and 533 fungal strains were isolated from diseased vines. Morphological, physiological and molecular (5.8S rRNA gene-ITS sequencing) analyses revealed that isolates belonged to 35 distinct fungal genera, including well-known (e.g., <i>Botryosphaeria</i> sp., <i>Diaporthe</i> spp., <i>Eutypa</i> sp., <i>Diplodia</i> sp., <i>Fomitiporia</i> sp., <i>Phaeoacremonium</i> spp., <i>Phaeomoniella</i> sp.) and lesser-known (e.g., <i>Neosetophoma</i> sp., <i>Seimatosporium</i> sp., <i>Didymosphaeria</i> sp., <i>Kalmusia</i> sp.) grapevine wood inhabitants. The GTDs-inducing population structure differed significantly among the discrete geographical zones. <i>Phaeomoniella chlamydospora</i> (26.62%, <i>n</i> = 70), <i>Diaporthe</i> spp. (18.25%, <i>n</i> = 48) and <i>F. mediterranea</i> (10.27%, <i>n</i> = 27) were the most prevalent in Heraklion, whereas <i>D. seriata</i>, <i>Alternaria</i> spp., <i>P. chlamydospora</i> and <i>Fusarium</i> spp. were predominant in Nemea (central Greece). In Amyntaio and Kavala (northern Greece), <i>D. seriata</i> was the most frequently isolated species (>50% frequency). Multi-genes (<i>rDNA-ITS</i>, <i>LSU</i>, <i>tef1-α</i>, <i>tub2</i>, <i>act</i>) sequencing of selected isolates, followed by pathogenicity tests, revealed that <i>Neosetophoma italica</i>, <i>Seimatosporium vitis</i>, <i>Didymosphaeria variabile</i> and <i>Kalmusia variispora</i> caused wood infection, with the former being the most virulent. To the best of our knowledge, this is the first report of <i>N. italica</i> associated with GTDs worldwide. This is also the first record of <i>K. variispora</i>, <i>S. vitis</i> and <i>D. variabile</i> associated with wood infection of grapevine in Greece. The potential associations of disease indices with vine age, cultivar, GTD-associated population structure and the prevailing meteorological conditions in different viticultural zones in Greece are presented and discussed.
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